紫草素侧链1'-OH酯化修饰相关CCLs和SATs基因的克隆及功能分析

Shikonin and its derivatives have significantly pharmacological activities but poor druggability, esterification modification of its 1'-OH of side chain is the key point to improve the druggability. Esterification method of chemical synthesis had many disadvantages including none stereoselectivity, toxic reaction process and so on, while biocatalysis and metabolic engineering techniques can be used to obtain shikonin 1'-OH esterified derivatives specifically, efficiently and environmental friendly. In this project, we intend to synthesize the methods of chemistry, molecular biology, bioinformatics and synthetic biology, through transcriptome and metabolome differential analysis of the shikonin-deficiency shikonin-proficient and shikonin-deficient cell lines from Arnebia euchroma (Royle) Johnst, digging shikonin 1'-OH esterification modification related CCLs and SATs genes and identifying their function in expression system of Escherichia coli in vitro. On the one hand, obtain different shikonin 1'-OH esterified derivatives specifically by constructing recombinant expression strains overexpressing different CCLs and SATs at the same time by biocatalysis; On the other hand, reveal the role of CCLs and SATs in the process of shikonin 1'-OH esterification modification by controlling the expression levels of CCLs and SATs in hairy root of A. euchroma, and obtain high-yielding hairy roots of specific shikonin 1'-OH esterified derivatives. This study provides a new target and means for directional control of shikonin 1'-OH esterification modification with great significance to improve the druggability of shikonin and its derivatives.

紫草素类化合物药理活性显著但难以成药，对其侧链1'-OH酯化修饰是改善其成药性的关键。化学合成酯化方法存在无立体选择性、反应过程有毒等弊端，利用生物催化和代谢工程技术可期高效环保地获得不同结构紫草素1'-OH酯化物。本项目拟综合化学、分子生物学、生物信息学和合成生物学方法，通过新疆紫草红色高产细胞系与白色低产细胞系的转录组和代谢组差异分析，挖掘紫草素侧链1'-OH酯化修饰相关CCLs和SATs基因，利用大肠杆菌体外表达系统对其进行功能鉴定。一方面构建同时表达CCLs和SATs的重组菌株，以生物催化方式获得不同结构紫草素1'-OH酯化物；另一方面调控新疆紫草毛状根中CCLs和SATs表达水平，揭示其在紫草素1'-OH酯化修饰中的作用，并在此基础上获得定向高产紫草素1'-OH特定酯化物的毛状根。此研究为定向调控紫草素侧链1'-OH酯化修饰提供了全新目标 靶标 ，对改善其成药性具有重要意义。

紫草素类化合物药理活性显著，但因其无选择性地损伤正常细胞而无法成药，紫草素1'-OH酯化修饰是改善其成药性的关键。本研究以定向调控紫草素侧链1'-OH酯化修饰获得特定紫草素侧链酯化物为目标，对紫草素1'-OH酯化相关酶CCLs及SATs基因进行克隆鉴定。项目按照计划书顺利执行，完成了拟定的目标。执行期间发表学术论文4篇，其中SCI1篇，获得授权专利1项，培养研究生2名。具体取得的主要工作研究进展、结果和影响如下：.1）本项目克隆得到7条新疆紫草CCL基因，完成了AeCCLs基因原核表达载体的构建及转化、表达的条件进行优化、完成了AeCCLs基因基因的体外功能验证；发现AeCCL5和AeCCL7均可催化4-香豆酸生成4-香豆酰-辅酶A，参与紫草素生物合成关键底物对羟基苯甲酸的生物合成途径。.2）本项目克隆得到4条新疆紫草SAT基因和4条硬紫草SAT基因，完成了AeSATs基因和LeSATs基因原核表达载体的构建及转化、原核表达的条件进行优化，获得了全部基因的可溶性蛋白，完成了AeSATs基因和LeSATs基因的体外功能验证，发现AeSAT1能够催化异戊酰紫草素和异丁酰紫草素的生成，为通过生物催化及代谢工程手段定向调控紫草素侧链1'-OH酯化修饰提供了全新靶标。.3）在转基因毛状根的研究方面，本项目同时尝试了干扰和过表达转基因毛状根的研究，为获得紫草素特定1’-OH酯化物高产的毛状根搭建了技术平台。获得了2条CYP76B74同源基因的基因干扰型毛状根，发现干扰CYP76B75与CYP76B100表达后可以促进紫草素合成。.4）本项目除了开展了酰基转移酶的筛选和功能验证的工作还克隆得到15条新疆紫草糖基转移酶基因，通过体外酶促发现5条可以不同程度地催化咖啡酸及迷迭香酸的糖基化反应的糖基转移酶。这是继PGT、SAT被鉴定后，新疆紫草中药效生物合成成分相关转移酶取得的新进展。.5）为更好的筛选紫草素合成相关基因，本项目完成了新疆紫草的全基因组测序、组装和注释，为后续更深入的开展相关研究奠定了基础。